Evaluation of 41 single nucleotide polymorphisms in canine diffuse large B-cell lymphomas using MassARRAY

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of lymphoma in dogs with a multicentric form. This study aimed to assemble 41 variants of the previously reported genes and to investigate these variants in canine DLBCL using the Agena MassARRAY platform. These variants were chosen based on the high prevalence observed in canine B- and T-cell lymphomas, their significance for target therapy, and compatibility for multiplex PCR amplification. Lymph node biopsy was performed from 60 dogs with B-cell lymphoma comprising 47 purebred and 13 crossbred dogs. All dogs presented single nucleotide polymorphisms (SNPs) at HYAL4 and SATB1 genes. The lesser mutual SNPs were observed at SEL1L, excluding a cocker spaniel, and c-Kit, with the exception of a pug and a French bulldog. Even though no statistical association was noted between each SNP and dog breed, purebreds were 3.88 times more likely to have a SNP at FLT3 rs852342480 (95%CI 0.50–45.03, p = 0.26), 3.64 times at TRAF3 F306X (95%CI 0.58–42.50, p = 0.43) and 2.66 times at TRAF3 E303EX (95%CI 0.56–13.12, p = 0.31). Also, DLBCL dogs (CHOP-based treatment) with c-Kit T425= had a poorer prognosis with shorter median overall survival times (OST) than dogs with the wild type. Dogs treated with COP chemotherapy and contained 3–5 variants at SEL1L were associated with decreased median OST. Therefore, this SNP’s lymphoma panel provides valuable information that we can use to outline a prognosis and develop a treatment plan for the targeted therapy of each dog.

Scientific Reports | (2022) 12:5120 | https://doi.org/10.1038/s41598-022-09112-0 www.nature.com/scientificreports/ Gene expression analysis and next-generation sequencing technology have been used to investigate the potential risk genes and probable signaling pathways that may cause specific diseases and cancers. In humans, global gene expression profiling has indicated two subtypes of DLBCL: neoplastic cells that derive from germinal center B cells (GCB DLBCL) or those from post-germinal activated B cells (ABC DLBCL) 13 . The ABC subgroup is notably stimulated through B-cell receptors including many of the NF-kB target genes 14 , whereas genetic mutations in GCB DLBCL are frequently observed in chromatin modifiers and histone proteins such as KMT2D, MYD88, CARD11, EZH2 and CREBBP 15,16 . Interestingly, Richards et al. 17 investigated gene expression profiling in canine B-cell lymphomas and found similarities to humans. Based on the immunohistochemistry and gene expression pattern, two distinct groups were classified as the ABC-like and the GCB-like DLBCL. The GCB-like group had higher expression of IRAK1BP1 and STAT4, while the ABC type had increased expression of NF-kB pathway genes. Furthermore, the canine ABC-like group had significantly poorer the progression-free and overall survival times compared to the GCB subgroup resembling with human DLBCL.
Several studies currently have used whole genome, whole exome and whole transcriptome (RNA-Seq) sequencing to explore the aberrant genes contributing to lymphomagenesis in specific dog breeds. Elvers et al. 18 investigated the genetic risk factors in three lymphoma-predisposed breeds: boxers for T cells, cocker spaniels for B cells and golden retrievers for B and T cells. The authors found strong similarities between the mutations in both dog breeds with B-cell lymphoma, occurring in TRAF3-MAP3K14, FBXW7 and POT1. However, the boxer with T-cell lymphoma typically had mutations in the PTEN-mTOR pathway, which was dissimilar to the golden retriever with T-cell lymphoma that usually exhibited mutations in genes related to cellular metabolism. In addition to these findings, multiple somatic point mutations were identified in canine B-cell lymphoma including TRAF3, POT1, LMNB1 and MVB12A [19][20][21] . Recently, single nucleotide polymorphisms (SNPs) that affected SPAM1, HYAL4, HYLAP1, PTEN and SATB1 were noted in canine T-cell lymphomas [22][23][24] .
Even though fresh frozen tissue is more preferable for genetic mutation analysis due to conserving DNA quality, formalin-fixed paraffin embedded (FFPE) technique is a standard method in routine work in the pathology unit. The FFPE tissue could preserve the cellular morphology and keep at room temperature for several years. However, the fixation process usually causes DNA cross-linkage, degradation, and fragmentation which could interfere the accuracy of molecular studies especially RNA 25 . There are several studies have compared the DNA quality between fresh and FFPE specimens by using next generation sequencing. The results of mutation analysis from those two types of samples were comparable 26 . Moreover, FFPE tissues from dogs with call rate > 65% provided similar results of single nucleotide variation when compared to fresh whole blood samples 27 .
In the present study, we assembled a custom SNP panel of the previously reported genes that may drive lymphomagenesis in dogs. The criteria for variants selection were high prevalence in specific dog breeds, application for target therapy, and primer compatibility for multiplex polymerase chain reaction (PCR) with maximum ability of MassARRAY. This lymphoma SNP panel was then investigated in dogs with DLBCL, studying 47 purebred and 13 crossbred dogs, from archival FFPE samples. Beyond SNPs' evaluation, the mutation genotyping panel was found to be different in each dog. This might be of relevance when it comes to outlining prognoses and selecting targeted therapies for affected dogs, by contributing toward increasing the treatment efficacy of personalized medicine.

Methods
Tissue samples and immunohistochemistry. Formalin-fixed paraffin-embedded (FFPE) tissues were obtained from the archive of the Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University and SQ Reference Lab, China between 2011 and 2021. A statement to confirm that all methods were carried out in accordance with relevant guidelines, regulations and sampling procedures was approved by the Chulalongkorn University Animal Care and Use Committee.
All dogs had a history of generalized lymphadenopathy and were diagnosed as having nodal lymphomas based on the cytopathology and histopathology. Lymph node samples were collected at the time of presentation and before receiving chemotherapy. Immunostaining with CD3, CD20 and Pax5 and/or a clonality test were performed for lymphoma subtype classification following REAL/WHO (Revised European American Lymphoma/World Health Organization) 1 . The immunohistochemistry for CD20, CD3 and Pax5 was adapted from a previous study 28 . Briefly, tissue section was quenched endogenous peroxidase by 0.3% (v/v) H 2 O 2 for 30 min and 5% bovine serum albumin was used for non-specific blocking for 20 min. Antigen was unmasked for CD3 and Pax5 by 10 mM citrate buffer (pH 6) in water bath at 95 °C for 20 min. Then, the slide was incubated with primary antibodies: CD20 (1:300, ab27093, Abcam, MA, USA), CD3 (1:10, PF. Moore, CA, USA), and Pax5 (1:100, 1EW, Leica, Newcastle Upon Tyne, UK) for 1.5 h. Secondary antibodies were applied for 30 min using EnVision system-HRP, mouse/rabbit (Dako, Glostrup, Denmark) for CD20 and Pax5 and ImmPRESS HRP, rat (Vector Laboratories Inc., CA, USA) for CD3. Visualization system for CD20 and Pax5 was DAB (Dako) and for CD3 was NovaRED (Vector Laboratories Inc.) as a substrate. Only DLBCL was included in this study. Demographic information was also recorded for each subject (Table 1). From 60 dogs, 26 dogs were treated with COP-based protocol (cyclophosphamide, vincristine, and prednisolone), 12 dogs received CHOP-based protocol, and the rest were loss of contact or death after diagnosis. DNA extraction. Each FFPE block was shaved to 75-100 µm thickness in a sterile microcentrifuge tube. The samples were then deparaffinized with xylene and absolute ethanol, respectively, before genomic DNA extraction using a DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) following manufacturer's instructions. The DNA concentration from each sample was quantified using a Nanodrop Lite Spectrophotometer (Thermo Scientific, MA, USA) and kept at -20 °C until use. The DNA samples were required to have a level of quality To eliminate excess dNTPs from the previous step, 0.17 μL of 10X SAP buffer, 0.3 μL of SAP enzyme and 1.53 μL of HPLC-grade H 2 O were added to the step-one PCR products, to a total volume of 7 μL, and incubated in the thermal cycles of 37 °C for 40 min following by 85 °C for 5 min. The final step of the single-base extension reaction was performed with an IPLEX® Pro Reagent Kit (Agena Bioscience) to hybridize and elongate the extension primers at the nucleotide position of interest. For the single-base extension (ddNTPs), 0.2 μL IPLEX® Buffer (10X), 0.2 μL IPLEX® Terminator Mix (10X), 0.04 μL IPLEX® Pro Enzyme (33 U/μL) and 0.94 μL extension primers (0.58-1.21 μM) were mixed with the step-two products, and H 2 O was added to a total volume of 9 μL. The reaction was performed at 95 °C for 30 s, followed by five cycles of 95 °C for 5 s, 52 °C for 5 s and 80 °C for 5 s, for a total of 40 cycles, with a final extension at 72 °C for 3 min.
For desalination, 29 μL HPLC-grade H 2 O and 13 μL clean resin (96-well microplates) were added to the step-three extension products. Afterward, the supernatant was spotted onto a matrix-precoated SpectroCHIP® through a MassARRAY Nanodispenser and scanned using a MassARRAY Analyzer. The results were analyzed using MassARRAY Typer Software (v.4.1.8.3). The mutation was distinguished with TOF (time-of-flight) mass spectrometry on the basis of different molecular weights. The peaks in the mass spectrum were identified as mutations. Only those samples with a success rate greater than 80% were included in the analysis. Genotyping calls were viewed in call cluster plots, and peak intensities were reviewed in each respective sample spectrum. A SIFT (Sorting Intolerant From Tolerant) score from variant effector predictor in dog genome database was used to predict whether an amino acid substitution affects protein function (https:// asia. ensem bl. org/ Multi/ Tools/ VEP).

Data analysis.
Kaplan-Meier survival analysis was used to analyze overall survival times (OST) from sex, breed, and chemotherapeutic protocol using GraphPad Prism version 9.2.0 (GraphPad Software, CA, USA). The SNP variants were selected to predict the OST and separately compared between the mutant and wild-type dogs received COP and CHOP. The p-value of survival analysis was calculated by medians of a log-rank test. The odds ratio of SNP location and dog breeds was calculated by Fisher's exact test. P < 0.05 was understood to represent statistical significance.

Discussion
Our study developed a SNP panel of 41 locations and investigated in canine nodal DLBCL using a MassAR-RAY system. The MassARRAY panel could evaluate up to 40 variants from 192 samples in a single run and it is affordable in relation to other technologies available. The prevailing variants in DLBCL were SATB1 Q420P (96.67%), c-Kit rs22299980 (75.86%) and SEL1L 8:53778185 (74.58%). Among three locations, SATB1 Q420P had a moderate impact on the protein function, with a SIFT score equal to 0.0 (deleterious). In contrast, a nonsense mutation of c-Kit had a low impact and an intron variant of SEL1L was a modifier. Special AT-rich sequence-binding protein 1 (SATB1) is a global transcription regulator and chromatin organizer. SATB1 encodes a binding nuclear matrix protein that recruits the chromatin remodeling factor, to regulate     www.nature.com/scientificreports/ golden retrievers 18 . Harris et al. 22 also found that one non-boxer dog with PTCL had SNP at c. 1259 T > G in SATB1 L420R . The author selected this SNP location followed aforementioned studies; however, different mutation (c. 1259 A > C) was used in this SNP panel. The polymorphisms of SATB1 Q420P, Q420PX were observed in all purebred and mixed-breed dogs with DLBCL. In human cancers, this gene promotes tumor progression and metastasis in breast cancer, colorectal cancer and cutaneous T-cell lymphoma [30][31][32] . As the authors could not indicate the significance of mutated SATB1 and its expression in high-grade nodal lymphomas in dogs, further study is required to confirm the significant pathogenesis, and a target drug that could restore SATB1 function might be helpful for the treatment of canine lymphomas. The c-Kit proto-oncogene is encoded as the transmembrane type-III receptor tyrosine kinase KIT, which is expressed in the hematopoietic progenitor of both myeloid and lymphoid cells. It plays a role in proliferation, cell survival and differentiation of hematopoietic precursors 33,34 . As c-Kit mutations are mainly reported in canine mast-cell tumors, few studies have investigated its mutation in canine leukemia and lymphoma. A Table 3. Survival analysis of selected mutant and wild type genes in 30 diffuse large B-cell lymphomas treated with COP-and CHOP-based chemotherapy. Significant values are in bold.   37 investigated the response rate of toceranib phosphate, one of the tyrosine kinase inhibitor (TKI) drugs used to treat unresectable high-grade mast-cell tumor with c-Kit mutation, in dogs with refractory T-cell lymphoma. The overall response rate of this monotherapy was only 40% in three dogs with partial remission and two dogs with stable disease. Therefore, the effectiveness and efficacy of TKI to treat canine lymphoma with c-Kit T425 = /T425TX require further investigation. In addition, c-Kit mutation has a significant prognosis in canine cutaneous mast cell tumors. Internal tandem duplication in exon 11 were approximately observed in 30-50% of higher grade mast cell tumors 38 . It has been associated with decreased survival times and progression-free survivals 39,40 . No evidence has reported on c-Kit mutation and its prognostic value in canine lymphoma. As such, its significance on prognosis needs further investigation in canine DLBCL. The common SNP locations identified in canine DLBCL were POT1 and TRAF3 18 . The telomere-binding protein protection of telomeres 1 (POT1), encoded by POT1, serves as providing telomere maintenance, and its dysfunction can lead to defective telomere replication, in turn, leading to genomic instability and enhanced carcinogenesis 41 . The adaptor protein TNF receptor associated factor 3 (TRAF3) is a tumor-suppressor gene that plays a critical role in B lymphocyte survival. A TRAF3 mutation leads to upregulation of the NF-kB pathway 19 . Both POT1 and TRAF3 mutations have been reported in human and canine B-cell lymphoma 18,19,21 . Elvers et al. 18 found POT1 and TRAF3 mutations in 17-20% of B-cell lymphoma cases in both golden retrievers and cocker spaniels. Therefore, our study selected SNP mutations of the POT1 and TRAF3 genes, following Smith et al. 21 . Each dog with a POT1 mutation tended to have more than one enriched variant, with a similar observation in TRAF3. Therefore, the novel target drugs against POT1 and TRAF3 might have therapeutic potential for treating DLBCL in dogs.
As lymphoma-risked genes depend on hereditary or somatic mutations, the best way to determine the significant genes is in one population, such as in a predisposing breed like boxers or retriever dogs. Our study focused on the application of the MassARRAY technique to detect nominated lymphoma variants from a valuable database in various dog breeds with the DLBCL subtype. Thus, it was possible that the candidate SNPs were not discovered in some dogs or were discovered at lower rates than usual. Moreover, few primers of each SNP location in our study had an amplification efficiency lower than 60%, especially in ZC3H7A H931R . Although the true prevalence could not be determined in our study, nine out of ten dogs (90%) had a mutation in ZC3H7A H931R . The missense mutation of ZC3H7A was also reported in canine DLBCL, with predictable functional consequences 20 .